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2
*
Research Institute of Child Nutrition, Dortmund, Germany;
Institute of Nutrition, University of Giessen, Giessen, Germany; and
**
Max-Planck-Institute for Molecular Physiology, Dortmund, Germany
2To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • milk oligosaccharides • digestion • humans • brush border membrane vesicles
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Rudloff and Kunz 1997
). There are
indications that human milk oligosaccharides
(HMO)3
could protect breast-fed infants against infections and
inflammations, an ability attributed to their structural similarities
to immunmodulating components and cell surface glycoconjugates
(Kunz and Rudloff 1993
, Wold and Hanson 1994
, Zopf and Roth 1996
). For example, it has
been shown that oligosaccharides can prevent bacterial adhesion to
intestinal cells (Newburg 1997
, Varki 1993
). Furthermore, they could function as prebiotics by
influencing the growth of a nonpathogenic microflora in the gut of
breast-fed infants. A prerequisite for these functions is that they
remain undigested in the small intestine. However, no studies focusing
on the digestibility of HMO by enzymes within the human
orogastrointestinal tract (GIT) have been published to date.
Furthermore, it is not known whether a low gastric pH has an effect on
these structures. It is widely thought that HMO are not digested
because no enzyme present in the GIT has been found to be capable of
cleaving fucose, N-acetyl-neuraminic acid (NeuAc) and
N-acetylglucosamine from oligosaccharides and
glycoconjugates. On the other hand, the alimentary tract of the neonate
contains several glycosidases such as amylase, maltase, lactase and
sucrase (Antonowicz et al. 1974
, Gray 1975
, Hamosh 1996
, Kien 1996
),
which have not yet been tested for their ability to cleave HMO.
Therefore, we investigated whether HMO are degraded by human salivary
amylase, porcine pancreatic amylase or brush border membrane vesicles
(BBMV) and whether HMO are resistant to a low pH similar to the one
prevailing in the infant’s stomach. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human milk samples were collected from six exclusively lactating, healthy mothers 3 wk after delivery between wk 28 and 32 of gestation. Oligosaccharide standards were purchased from Dextra (London, UK), lactose and lactulose, porcine pancreatic amylase (A 6255) and human salivary amylase (A 0521) from Sigma (Deisenhofen, Germany). Porcine small intestine was obtained from a local slaughterhouse immediately after veterinary inspection. The intestine was rinsed with ice-cold HEPES-saline buffer (10 mmol/L HEPES, 150 mmol/L NaCl), pH 7.1, and placed into ice-cold HEPES-saline buffer for transportation.
Preparation of HMO.
HMO were isolated as previously described with few modifications
(Kunz et al. 1996
). Human milk was centrifuged at 3000
x g at 4°C for 20 min, the lipid layer removed
and the aqueous phase decanted and filtered through glass wool. Of
precooled 95% ethanol, 66% (v/v) was added to precipitate the
protein. The solution was stirred gently on ice for 3 h,
centrifuged at 10,500 x g at 4°C for 1 h
and the supernatant was freeze-dried. To remove lactose and
monosaccharides, 440 mg of the freeze-dried supernatant was
dissolved in 4 mL H2O, filtered through a
0.45-µm filter (Nalgene 190-2545, Rochester, NY, USA)
and applied to a Sephadex G25 column (100 x 2.6 cm i.d.,
Pharmacia Biotech, Uppsala, Sweden). The eluent was water (flow
rate 2 mL/min). The eluate was collected in 10-mL fractions, and the
absorption was measured at 195 nm and 280 nm.
Carbohydrate-containing fractions, which were stained with orcinol
for carbohydrates or ninhydrin for protein (Kunz et al. 1996
, Rudloff et al. 1996
), were characterized
by high pH anion-exchange chromatography with pulsed amperometric
detection (HPAEC-PAD) and silica-high performance TLC (HPTLC).
Ninhydrin-positive fractions, which also contained
oligosaccharides, were prepared separately. HMO were separated into
neutral and acidic fractions using fast protein liquid chromatography
anion-exchange (Resource Q; 6-mL volume; Pharmacia Biotech,
Uppsala, Sweden). The freeze-dried oligosaccharides from each
Sephadex G25 run were dissolved in 1 mL H2O, filtered
through a 0.2-µm filter (Nalgene 0.2 190-2520) and
applied to a Resource Q column using eluent A (H2O) from 0
to 7.5 min, followed by a gradient up to 55% eluent B (0.6 mol/L NaCl)
in 42.5 min with a flow rate of 2 mL/min. Finally, eluent B was run up
to 100% within 2 min. Eluting fractions were monitored at 214 and 280
nm for residual proteins. The acidic fractions were desalted on a
Sephadex G25 column (100 x 2.5 cm i.d.). The carbohydrates were
detected at 195 nm and by HPAEC-PAD. Salt concentration was
determined by atomic absorption spectrometry.
Preparation of intestinal BBMV.
BBMV were prepared by the differential precipitation method according
to Booth and Kenny (1974)
. After the first 20 cm and the
last 50 cm were discarded, the intestine was washed several times with
ice-cold HEPES-saline buffer, pH 7.1, then slit open and cut into
20-cm long pieces, which were rinsed again with buffer. All steps were
carried out at 4°C. The mucosa were gently scraped off the underlying
muscular tissue by using a glass slide; 
5 g of mucosal tissue (wet
weight) was homogenized in 35 mL of buffer consisting of 50 mmol/L
mannitol and 2 mmol/L Tris-HCl, pH 7.1, using a Waring blender for
30 s at full speed, followed by a 1-min interval and then an
additional 30 s blending. MgCl2 was added to a final
concentration of 10 mmol/L. After 15 min, the homogenate was
centrifuged at 1400 x g for 12 min; the
supernatant was collected and then centrifuged at 16,000 x g for 20 min. The pellet was taken up in PBS, pH 7.0,
and homogenized in a loose-fitting glass-Teflon homogenizer at low
speed. After the final centrifugation at 16,000 x g for 20 min, the resulting pellet was resuspended in 50
µL PBS by repeated suction through a 26-gauge needle
and stored at -80°C.
The efficiency of the preparation was checked by determining the
activity of the marker enzymes sucrase, lactase, maltase and
isomaltase, i.e., the liberation of glucose during the incubation
of the BBMV with sucrose, lactose, maltose and isomaltose. We incubated
100 µL of each oligosaccharide solution (56 mmol/L)
buffered by PBS (pH 6.5) for 0 (blank) or 30 min with 10
µL (for maltase assay, only 5 µL)
BBMV solution. To test the stability of the enzyme activity within the
BBMV, sucrase was chosen as marker enzyme. Therefore, we incubated 10
µL BBMV for 2, 4 and 24 h in 100
µL PBS (pH 7.0) at 37°C and then assayed the enzyme
activity as described above. The amount of glucose liberated within the
incubation time was detected by the hexokinase/glucose-6-phosphate
dehydrogenase procedure (Bergmeyer et al. 1974
) using a
test kit from Boehringer (Mannheim, Germany). The activity of the
disaccharidases was expressed in units, where 1 U was
equivalent to 1 µmol substrate hydrolyzed per min at
37°C.The specific activity of marker enzymes per milligram protein
was determined according to the method of Lowry et al. (1951)
. Furthermore, the purity of the preparation was
checked by assaying the activities of acidic phosphatase as a marker
enzyme for lysosomes and alkaline phosphatase for the apical brush
border membrane (BBM) using p-nitrophenyl phosphate as
the substrate (Bergmeyer 1983 et al.
). In addition, 1
mmol/L L-p-bromotetramisole was added to the
acidic phosphatase assay to inhibit the residual alkaline phosphatase
activity at pH 4.5 (Borgers and Thoné 1975
).
Determination of the enzyme kinetics.
To determine the enzyme kinetics of the intestinal disaccharidases, the enzyme activity was measured at various oligosaccharide concentrations between 10 and 55 mmol/L and the glucose content measured as described for the disaccharidase assays.
Digestion studies.
All digestion assays were performed on each of the six neutral and six acidic HMO fractions with each enzyme and all assays were run at least twice.
Oligosaccharide solution for digestion experiments.
For all digestion experiments, 0.5 mg of the freeze-dried HMO dissolved in 100 µL PBS (pH 7.0) was used. The standard milk oligosaccharides, lactulose and lactose, had a concentration of 1 g/L PBS (pH 7.0).
General digestion procedure.
Oligosaccharide solution (100 µL) was incubated at 37°C for 2 h with each of the enzymes and the BBMV or PBS as control. The reaction of the digestion experiments was stopped by the addition of 100 µL 10% trichloroacetic acid. The solution was centrifuged at 2600 x g for 5 min (Hettich Universal 30 F centrifuge, rotor type 1412, Tuttlingen, Germany). The supernatant was adjusted to pH 7.0 using NaOH (0.5 mol/L) and then freeze-dried.
Studies on the digestibility of HMO using human salivary amylase.
Before the enzyme was used, its activity was tested on maltodextrin. The oligosaccharide solutions were incubated for 1 min with 1 U salivary amylase at pH 7.0 and 5.5. For the latter assays, the pH was adjusted with HCl (0.2 mol/L) to 5.5 after the first incubation step and then further incubated at 37°C for 2 h.
Influence of low gastric pH of the stomach on the milk oligosaccharide composition.
HMO solutions were adjusted to pH 2.5 using HCl (0.2 mol/L) and incubated as described above. The pH was then readjusted to pH 7.0 with NaHCO3 (0.2 mol/L).
Studies on the digestibility of HMO by a porcine pancreatic amylase.
Before using the amylase, the activity of the enzyme was tested on maltodextrin. For digestion assays, 100 U pancreatic amylase was added to the HMO solutions and incubated.
Digestibility of HMO using BBMV.
BBMV (10 µL) were added to the HMO or standard milk oligosaccharide solution and incubated for up to 24 h. To test the influence of the pH on the digestibility of HMO, we used PBS buffer ranging in pH from 5.0 to 7.0. The reaction was terminated by heating the samples at 95°C for 2 min followed by the stop procedure as described above.
Digestibility of oligosaccharide standards, lactulose and lactose using BBMV.
For the digestion studies of the standard oligosaccharides, lactulose and lactose, oligosaccharide solutions were incubated with 10 µL BBMV. The reaction was terminated by heating at 95°C for 2 min followed by the normal stop procedure as described above. For the inhibition studies, Tris-buffer (37.5, 75 and 150 mmol/L) was added to the digestion assay and the pH adjusted to pH 6.5.
Quantification of oligosaccharides.
Aliquots of the oligosaccharide solutions and the digestion assays
(5–10 µL) were characterized by HPAEC-PAD and by
silica-HPTLC as described previously (Kunz et al. 1996
). The peak areas of the BBMV digestion assays were highly
reproducible and varied by <5% (n = 6). For
silica-HPTLC, 3-3.5 µL were used for each run.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using the described oligosaccharide preparation techniques, the complex
HMO were separated from monosaccharides, lactose, fat and proteins. The
HMO were then separated into neutral and acidic fractions by
anion-exchange chromatography (Fig. 1
). Little overlapping of the fractions was detected by HPAEC-PAD
(Fig. 1)
.
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During a 1-min incubation, human salivary amylase did not degrade
components in the neutral or acidic HMO fraction. Because salivary
amylase remains active in the stomach for long periods of time
(Hodge 1983
), we also tested the combination of salivary
amylase digestion at 37°C and pH 5.5 for an incubation period of
2 h. However, no effect was observed on either of the
oligosaccharide fractions (Fig. 2A
and
B). Comparable to this, porcine pancreatic amylase was also not capable
of cleaving components present in either the neutral or the acidic HMO
fraction (data not shown).
|
The neutral HMO were not hydrolyzed after a 2-h incubation at pH 2.5
and 37°C (Fig. 3A
). The acidic fractions, however, showed minor changes in their
oligosaccharide composition compared with the initial fraction by TLC
(silica-HPTLC) (see lactose band in Fig. 3B
and Fig. 4
). These effects were more pronounced when the pH was lowered to 2.0
(data not shown). As a result of the cleavage of acidic
oligosaccharides, free NeuAc (peak 3) increased slightly and a few new
neutral components (retention time < 17 min), in particular
lactose (peak 1), appeared (Fig. 4)
. Furthermore, there was a minor
decrease in the peak heights of all acidic components (peaks 2 and
4–7).
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The specific activities of BBM enzymes were enriched to various extents
(Table 1
), e.g., by a factor of 4.6 ± 2.7 for lactase and 10.6 ± 6.3
for alkaline phosphatase compared with the initial homogenate. The
yields of these enzymes were 6.3 ± 3.8% for lactase and 21.4
± 7.7% for alkaline phosphatase measured as a percentage of the
activity originally found in the homogenate. Even after the addition of
1 mmol/L of the alkaline phosphatase inhibitor
p-bromotetramisole, the enzyme remained active to a small
degree at pH 4.5. For membrane-bound sucrase, a
KM of 25.3 ± 11 mmol/L was
determined and for maltase a KM of
11.4 ± 4.3 mmol/L. After a 24-h incubation of the BBMV at 37°C
in PBS (pH 7.0), 85% of the initial sucrase activity was present.
Within the BBMV preparation, only minor amounts of the lysosomal marker
enzyme acid phosphatase were found (Table 1)
.
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12% of a lactulose standard was cleaved. The degradation of
lactulose and lactose was completely inhibited by the addition of Tris
(150 mmol/L), an inhibitor of intestinal disaccharidases.
After the 2-h incubation of the HMO with BBMV, slight modifications
were detected. The new components observed within the neutral fraction
after a 2-h incubation period were characteristic of glucose and
galactose, which have an identical retention time (peak 1, Fig. 5
). This was the result of the breakdown of small amounts of lactose
still present in the HMO fractions (peak 3 in Fig. 5
and Fig. 6A
). To test whether a longer incubation time had any effect, digestion
studies were performed for up to 24 h. By using silica-HPTLC,
one new component, which migrated below 3-fucosyl-lactose (see arrow
Fig. 7A
), was detected within the neutral oligosaccharide fractions in addition
to lactose, galactose and glucose. This component was present after a
4-h incubation. Using several oligosaccharide standards, we were able
to show that the enzymes within the BBMV were able to cleave
nonfucosylated oligosaccharides such as lacto-N-tetraose
(LNT) and neo-LNT (lane 4 in Fig. 7B
) but not those that
were fucosylated such as lacto-N-fucopentaose (LNFP) I and
LNFP III. The new component described above was identified as
lacto-N-triose (LN-Tri) by fast atom bombardment mass
spectrometry analysis. Furthermore, small amounts of lactose and
galactose were detected.
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). In terms of the whole acidic fraction, these components accounted for
<5%. In contrast to the neutral fraction, the amount of new
components did not increase within an incubation period of up to
24 h. Furthermore, a pH in the range between 5.0 and 7.0 had no
effect on the digestibility of the HMO by the BBMV. The small changes
could be detected only by HPAEC-PAD (Fig. 8
) and not by
silica-HPTLC (Fig. 6B
).
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Due to a lack of commercially available glycosidases derived from the
human GIT, we used porcine intestinal enzymes and BBMV because of their
structural and developmental similarities to the human GIT
(Moughan et al. 1992
, Tumbleson and Schook 1996
, Table 2
).
|
, Köstlin and Rauch 1957
, Turner et al. 1954
, Watson et al. 1972
). Salivary amylase is active within the pH range from 5 to
6 (Hamosh 1996
, Hodge 1983
). Therefore,
the salivary amylase digestion studies were performed at pH 5.5, which
is within the physiologic range of pH in the infant’s stomach
(Agunod et al. 1969
, Mason 1962
). Neither
human salivary amylase nor porcine pancreatic amylase degraded the
oligosaccharides present in human milk. Apart from the similarities
described in Table 2
, porcine and human pancreatic amylase have a
homology of 87% in their amino acid sequence (Swiss Prot database;
internet address: www.expasy.ch/sprot). Thus, we conclude that our
results obtained with porcine pancreatic amylase are also applicable to
the situation prevailing in the human gut.
The small changes that were observed after a 2-h incubation with acidic
HMO at pH 2.5, which is a physiologic pH for the gastric fluid of the
infant’s stomach (Halpern et al. 1992
, Heacock et al. 1992
), resulted mainly from the cleavage of NeuAc from
NeuAc-lactose, which explains the occurrence of lactose. However,
these changes represent <5%.
Another reason for using BBMV preparations in the digestion studies was
to avoid interference by intracellular enzymes, in particular lysosomal
enzymes such as a ß-galactosidase, neuraminidase (Ghosh et al. 1968
) and fucosidase, which might degrade HMO-like
structures. This was ensured by using the differential precipitation
method of Booth and Kenny (1974)
, which selectively
enriches the apical BBM. For the lysosomal marker enzyme acidic
phosphatase, only a low enrichment factor was found (Table 1)
. We
therefore conclude that the small changes seen after the digestion of
HMO with BBMV are not due to the activity of lysosomal enzymes.
Because we found minor changes within both HMO fractions after the 2-h incubation with BBMV, we tried to enhance these by a prolonged incubation time. The amount of newly appearing components within the neutral, but not within the acidic fraction were increased. Thus, we conclude that the component(s) that were cleaved by BBMV within the acidic fraction were present in very small amounts. Furthermore, the fact that only nonfucosylated and nonsialyated oligosaccharides were digested is an indication that fucose and NeuAc prevent the degradation of the oligosaccharides within the small intestine.
Finally, we conclude that within the physiologic range of
incubation time, pH and enzyme activity, a substantial cleavage of HMO
does not occur. Our findings are supported by studies in which
HMO-like structures were detected in urine and feces of
breast-fed infants (Kunz et al. 2000
,
Obermeier et al. 1999
, Rudloff et al. 1996
, Sabharwal et al. 1988
). Therefore, because
HMO remain undigested, one of the prerequisites required to function as
prebiotics by influencing the growth of a bifidus flora has been
fulfilled. In addition, they have the potential to exert local
anti-inflammatory effects within the intestine of breast-fed
infants (Kunz et al. 2000
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BBM, brush border membrane;
BBMV, brush border membrane vesicles; GIT, orogastrointestinal tract;
HMO, human milk oligosaccharides; HPAEC-PAD, high pH
anion-exchange chromatography with pulsed amperometric detection;
LNFP, lacto-N-fucopentaose; LNT,
lacto-N-tetraose; LNTri, lacto-N-triose;
neo-LNT, neo-lacto-N-tetraose; NeuAc,
N-acetyl-neuraminic acid; silica-HPTLC, high
performance TLC.
Manuscript received June 5, 2000. Initial review completed July 20, 2000. Revision accepted August 31, 2000.
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